Title

Author

Date of Award

2012

Document Type

Thesis

RU Laboratory

Fischetti Laboratory

Abstract

Bacteriophage-based diagnostics and therapeutics have been recognized as tools to combat bacterial infections for nearly a century. Wip1 is a recently isolated phage that infects Bacillus anthracis, the notorious biothreat pathogen and gram-positive bacterium that causes anthrax disease. The current standard for identifying suspected B. anthracis involves testing for WÎ³ phage sensitivity. However, studies have shown that the narrow Wip1 host range is even more specific to B. anthracis than that of WÎ³, suggesting that Wip1 may be a superior diagnostic tool. Wip1â€™s high specificity to B. anthracis is likely mediated by the initial recognition and binding of the virus to the host cell. Most bacteriophages interact with bacterial surfaces using tail fibers, but Wip1 is a tailless phage possessing an icosahedral protein coat covering an internal lipid membrane. These features indicate that Wip1 belongs to the family Tectiviridae, a relatively rare phage group with a proposed evolutionary lineage to the mammalian adenovirus. Tectiviruses possess protruding spike complexes at their vertices that are responsible for host recognition and binding. Based on adsorption assays, we confirmed that Wip1â€™s host range specificity is mediated by its receptor-binding ligand. In order to predict candidate gene products for the Wip1 spike complex, we sequenced the Wip1 genome and conducted extensive genomic analyses with other tectiviral genomes. Observations from subsequent recombinant Wip1 protein expression and purification indicated that Wip1 p23 and p24 form a stable complex. We identified the unique protein Wip1 p23 as a receptor-binding protein using competitive inhibition and antibody neutralization assays. From indirect immunofluorescence microscopy, we also demonstrated that Wip1 p23 binds very specifically to the surface of B. anthracis. Finally, we characterized the bacterial receptor for Wip1 as a Sap-dependent cell wall component. The work in this dissertation has produced a significantly more detailed understanding of Wip1â€™s highly specific tropism for B. anthracis. The findings that we report here also demonstrate that Wip1 phage and its receptor-binding ligand p23 are promising, new tools in the detection of B. anthracis.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.